Ricin chromatography

Barbieri, L., and Stirpe, F. (1982) Dye affinity chromatography of ricin subunits. Cancer Surv. 1, 489-520. 

Protein toxins such as botulism, staphylococcal enterotoxin B, or ricin can be separated with gas or liquid chromatography, electrophoresis, or a combination. The pChemLab (Sandia National Laboratories Albuquerque, NM) series of instruments includes a hand-held Bio Detector. Proteins in the sample are labeled with fluorescent tags, and nanoliter volumes of samples are separated by microchannels etched into a glass chip. The separation occurs as the sample moves through the channels and identification is based on retention times. The analyses can be completed within 10 min. 

Multiple purification-schemes have been applied to the separation of the toxin and the hemagglutinin.144,146,147,150,194,64,-651 Fractionation using salt and ethanol precipitation led to crystallization844,645 of the toxin known as ricin or ricin D. The hemagglutinin was isolated, free from toxic activity, by ion-exchange chromatography and gel filtration.642,646-648 With the introduction of affinity chromatography on Sepharose 4B, to which both proteins bind, purification of the two R. 

Analytical methods include immune methods (ELISA) and liquid chromatography/mass spectrometry (LC/MS). Gastric contents can be assayed and ricin can be detected in blood and body fluids by radioimmunoassay and LC/MS (Darby et al, 2001 Mouser et al, 2007). 

Mouser, P., Filigenzi, M.S., Puschner, B. et al. (2007). Fatal ricin toxicosis in a puppy confirmed by liquid chromatography/mass spectrometry when using ricinine as a marker. J. Vet. Diagn. Invest 19 216-20. 

In the case of ricin A chain, a proportion of unreacted RIP binds irreversibly to the S-200 column during the final purification. Tlie column can be cleaned by washing with O.lMNaOH before further chromatography. 

Here we report the results obtained using MIANS-labeling in conjunction with affinity chromatography to map free thiols in reduced, native ricin A-chain. We further describe the results obtained when utilizing this method to isolate ligand-bound ricin B-chain peptides. 

Following affinity chromatography of the MIANS-ricin A-chain digest over the anti-MIANS column, the eluted fraction was run over the C,g RP-HPLC column utilizing gradient elution conditions. Individual peptides were collected for amino acid analysis (data not shown) and for sequencing. 

Hines, H.B., Brueggemann, E.E. and Hale, M.L. (2004) High-performance hquid chromatography-mass selective detection assay for adenine released from a synthetic RNA substrate by ricin A chain. Anal Biochem, 330, 119-122. 

We calculated the relative RT of the molecular species of PC from the six [ CjFA incubations based on the RT (32.15 min) of l-palmitoyl-2-oleoyl-PC in our previous report (6) by co-chromatography with this PC standard (unpublished). The calculated RT of the six 2-ricinoleoyl-PC in Figure 3B from the [ C]ricinoleate incubation are 9.4 min (1,2-ricin-oleoyl-PC), 14.6 min (l-linolenoyl-2-ricinoleoyl-PC), 16.9 min (l-linoleoyl-2-ricinoleoyl-PC), 18.5 min (1-palmitoyl-... 

Fig. 3. The C8 high-performance liquid chromatography (HPLC) radiochromatogram for the separation of molecular species of phosphatidylcholine (PC see Fig. 1,2 pL from 100 il methanol solution of PC and ricin-noleoyl-PC combined) from the castor microsomal incubation of [ " C]-oleic acid co-chromatographed with standard 1-palmitoyl-2-oleoyl-PC (For HPLC conditions, see Experimental Procedures.) (1) 1-ricinoleoyl-2-oleoyl-PC, 19.7 min (2) 1-linolenoyl-2-oleoyl-PC, 27.8 min (3) 1-linoleoyl-2-oleoyl-PC, 30.5 min (4) 1-palmitoyl-2-oleoyl-PC, 32.0 min (5) 1,2-dioleoyl-PC, 33.5 min (6) 1-stearoyl-2-oleoyl-PC, 36.3 min (7) 1-linolenoyl-2-ricinoleoyl-PC, 14.7 min (8) 1-linoleoyl-2-ricinoleoyl-PC, 16.9 min (9) 1-palmitoyl-2-ricinoleoyl-PC, 18.5 min (10) 1-stearoyl-2-ricinoleoyl-PC, 22.7 min (11) 1-linolenoyl-2-linoleoyl-PC, 25.0 min (12) 1-palmitoyl-2-linoleoyl-PC, 29.3 min.

Source Ler, S.G., Lee, F.K., Gopalakrishnakone, P. (2006) Trends in detection of warfare agents detection methods for ricin, staphylococcal enterotoxin B and T-2 toxin. Journal of Chromatography A, 1133,1-12. 

Becher, E, et ai. (2007) Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry. Analytical Chemistry, 79, 659-665. 

The detection of ricin directly by instrumental methods has been limited to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) detection (Darby et al., 2001 Brinkworth et al., 2009). Darby et al. (2001) separated ricin from other material present in samples using size exclusion chromatography and then hydrolyzed the isolated ricin with trypsin. The hydrolysate was analyzed by MALDI-TOF MS and ricin peptides identified by comparison of mass ions with an online database and comparison of identified peptides with those from a reference sample obtained from ricin. Brinkworth et al. (2009) generated and measured the intact ricin ion as opposed to peptide fragments. Since the presence of readily ionized substances such as salts in the sample can overwhelm the detection of ionized ricin, the sample was concentrated and ultrafiltered to eliminate material of MW below 30kDa. The method detected ricin at levels of 4 gg/mL. 

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